With the introduction of combination/highly active antiretroviral therapy (cART or HAART) in 1996, the diagnosis of HIV/AIDS is no longer a death sentence. However, due to the existence of latent pools of HIV infected cells (“latent reservoirs,” or “LR”) in patients on HAART, HIV viral levels rapidly rebound upon cessation of treatment. Thus, HIV infected individuals must commit to lifelong adherence to HAART regimens, with an estimated cost of over half a million dollars per individual. Significant government and industry resources have been deployed to generate a cure for HIV/AIDS. Formed in 1986, the National Institutes of Health, Division of AIDS (DAIDS) was tasked with a national research agenda to end the HIV/AIDS epidemic. DAIDS supports a global research portfolio on HIV/AIDS, related co-infections, and co-morbidities. The goal is to create an AIDS-free Generation, through innovative approaches aimed at: 1) halting the spread of HIV through effective and acceptable prevention strategies and a preventive vaccine; 2) treating and curing HIV infection; 3) establishing treatment and prevention strategies for the HIV co-infections and co-morbidities of greatest significance; and 4) partnering with scientific and community stakeholders to implement effective interventions.” If a cure becomes available, it will transform the lives of HIV-infected individuals and the financial landscape of this devastating disease. The ability to precisely and accurately measure low levels of HIV in latent reservoirs is a critical bottleneck in achieving a cure for HIV/AIDS.
Latent reservoirs consist of resting or memory CD4+ cells and other cells carrying, for example, the HIV-1 viral genome, either as pre-integration plasmids or integrated into relatively inactive regions of the DNA of CD4+ and potentially other host cells, as well as HIV-infected cells in HAART-inaccessible regions of the body. HIV-1 latent reservoirs include peripheral blood, lymph nodes (B cell follicles), gut-associated lymphatic tissue (GALT), central nervous system (brain and spinal cord), and oral mucosa.
Resting memory CD4+ T cells, typically sampled from peripheral blood mononuclear cells (PBMC), are a major component of the HIV-1 latent reservoir and stand as a major barrier to curing HIV-1 infection. While a variety of PCR- and culture-based assays have been developed to measure the size of the peripheral blood LR, there is little agreement between different assay results and no available assay appears to provide an accurate measurement of reservoir size. The lack of an accepted standard assay has remained a significant impediment in clinical trials seeking to evaluate novel HIV-1 eradication strategies. Well-validated, high-throughput assays that accurately quantitate latent reservoirs are urgently needed to assess complete eradication of HIV. Currently, there are no commercially or noncommercially available assays that adequately answer this need and which can be translated to widespread use.
The oral mucosa is a HAART-resistant HIV-1 latent reservoir (LR), which, unlike systemic immunity, is not restored to full immune competence by HAART treatment. The oral mucosa LR appears to include not only the expected CD4+ T cells, but also dendritic cells under constant reactivation by oral microbes and endotoxin. Further, oral epithelial cells, such as keratinocytes, may be susceptible to HIV infection and contribute to the oral HIV LR. The oral cavity is highly accessible for sequential noninvasive sampling and can routinely be sampled in outpatient or remote areas where resources are limited. A point-of-care assay that accurately and precisely quantifies the HIV-1 LR, along with corresponding viral output, from oral mucosa test samples would greatly facilitate development and dispensation of a cure for HIV AIDS.
The challenges in developing a quantitative diagnostic for the HIV latent reservoir are several: (1) low levels of HIV RNAs produced by latently infected cells; (2) variable expression of HIV RNAs produced by latently infected cells; (3) low levels of latently infected cells; and (4) the lack of reliable sampling methods for measuring HIV RNA levels from anatomical locations most relevant to eradicating the latent reservoir.
The quantitative ligation detection reaction (qLDR) technology provides a significantly improved molecular assay to accurately and precisely quantitate target RNA sequences and target RNA-producing cells, such as those present in latent HIV reservoirs. In an exemplary embodiment applying qLDR technology, latent HIV-1 RNA cells present in HIV-1 infected individuals on HAART are quantitated by accurately and precisely detecting spliced HIV mRNA directly from lysed CD4+-enriched peripheral mononuclear cells and oral mucosal cells.
qLDR employs a fluorogenic chemical autoligation reaction template by specific RNA sequences. In the closest prior art, a similar fluorogenic autoligation reaction provides detection of DNA G-quaduplexes and DNA single-nucleotide polymorphisms (Koripelly et al., 2010; Meguellati et al., 2010, 2013). However, fluorogenic autoligation detection reactions have not been developed for RNA spliced sites, RNA secondary structure, linear RNA sequences, nor for cell-based viral RNA. Autoligation detection reactions for RNA targets are in particular demand due a dearth of RNA-templated enzymatic reactions. While DNA-targeted fluorogenic autoligation detection has been used for limited testing of highly pure, artificial DNA constructs (Koripelly et al., 2010; Meguellati et al., 2010, 2013), RNA-targeted fluorogenic autoligation detection has not been developed and, after the present developments, has great promise for in vitro diagnostics. Beyond the difference in target (RNA vs. DNA), adaptation of autoligation reactions to RNA detection entails the formation of different probe-target structures which alter the chemistry of the reaction, different spacing between probes on the target sequence, the ability to detect significantly lower concentrations of target sequences in complex environments, the amount of variation in target tolerated by the probes, the ability to perform the reaction with and without denaturing and/or in isothermal or thermocycling conditions, the use of probe backbones that favor RNA binding, and the ability to use a much wider range of nonfluorescent reactive probe moieties to form a much wider range of fluorescent dyes for better detection and multiplex detection of multiple RNA targets.
In the past, direct detection of RNA has been difficult to achieve except by the use of hybridizing probes, which entail lengthy hybridization periods and multiple wash steps, followed by visualization procedures. There is a lack of RNA-specific enzymes similar to those used with DNA that achieve PCR amplification, single-nucleotide polymorphism (SNP) detection, and detection of specific RNA sequences and secondary structures. Thus, new methods for detecting RNA targets would benefit technology.